Evidence from many laboratories indicates that alpha-synuclein is implicated at the levels of both genetics and biochemical pathology in Parkinson's disease, dementia with Lewy bodies, multiple systems atroohy and other alpha-synucleinopathies. In Project 2, two investigators who have collaborated productively during the current Udall grant period will pursue the central hypothesis that certain post-translational modifications -- including glycan-association, ubiquitination and oligomerization -- of alpha-synuclein (alphaS) may represent key pathogenic steps in both familial and "sporadic" forms of PD. Based on substantial progress on this hypothesis during the current grant period and extensive preliminary data, we propose two broad, interrelated Specific Aims that apply a range of methods in cell biology, biochemistry and animal modeling to the questions of how the normally soluble alphaS protein is converted into metastable oligomers and higher polymers and whether this pathological conversion arises from its normal properties or via a distinct toxic pathway. Our Specific Aims include: (1) to pursue our novel findings that physiological alterations in neuronal fatty acid metabolism, particularly in the levels of long-chain PUFAs, help regulate the oligomerization state of aS, and that this effect involves an interaction between PUFAs and alphaS that occurs normally but is exaggerated in PD, DLB and other disorders in which alphaS oligomerizes progressively; and (2) to characterize two distinct events in alphaS processing that we have identified in human tissue and model them in dopaminergic cells: a) the glycan association of alphaS that enables its binding to Parkin, and b) the exocytosis of alphaS. We are strongly committed to completing the structural and glycobiological analysis of aSp22 (which we have now purified to homogeneity by lectin and antibody affinity steps) and fully characterizing the non-covalent association of alphaS with complex glycans that we have found. As part of this second Aim, we will also study the potentially related mechanism of alphaS exocytotic release into extracellular fluids that we have detected in humans in vivo. These and related experiments detailed herein should provide information about distinct post-translational modifications that may enhance the accumulation and aggregation of human alphaS into potentially neurotoxic oligomers in PD and other synucleinopathies.